Viroporin-like activity of the hairpin transmembrane domain of African swine fever virus B169L protein

Authors: Gladue, Douglas; GOMEZ-LUCAS, LIDIA - Consejo Superior De Investigaciones Cientificas (CSIC); LARGO, ENEKO - University Of The Basque Country Upv/ehu; Ramirez Medina, Elizabeth; TORRALBA, JOHANA - Consejo Superior De Investigaciones Cientificas (CSIC); QUERALT, MARIA - Consejo Superior De Investigaciones Cientificas (CSIC); ALCARAZ, ANTONIO - Jaume I University; Velazquez Salinas, Lauro; NIEVA, JOSE - Consejo Superior De Investigaciones Cientificas (CSIC); Borca, Manuel

Submitted to: Journal of Virology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/21/2024
Publication Date: 7/9/2024
Citation: Gladue, D.P., Gomez-Lucas, L., Largo, E., Ramirez Medina, E., Torralba, J., Queralt, M., Alcaraz, A., Velazquez Salinas, L., Nieva, J., Borca, M.V. 2024. Viroporin-like activity of the hairpin transmembrane domain of African swine fever virus B169L protein. Journal of Virology. 98(8). https://doi.org/10.1128/jvi.00231-24.
DOI: https://doi.org/10.1128/jvi.00231-24

Interpretive Summary: African swine fever, a devastating disease affecting domestic swine, is widely spread in Eurasia producing significant economic problems in the pork industry. Approaches to prevent/cure the disease is mainly restricted to the limited information concerning the role of most of the genes encoded by the large DNA virus genome. In this report, we present the experimental data on the functional characterization of the one gene called B117L. Data presented here indicates that the B117L gene encodes for a potentially essential protein associated with the virus membrane that could function as a viroporin, a type of protein that facilitates virus release from the cells.

Technical Abstract: African swine fever virus (ASFV), the causative agent of a contagious disease affecting wild and domestic swine, is a highly structural complex double stranded DNA virus. ASF is currently affecting pork production in a large geographical area including Eurasia and the Caribbean region. ASFV has a large genome which harbors more than 160 genes although the function of most of those genes have not been experimentally characterized. The function of B169L, identified in the ASFV proteome as a potential integral membrane protein, remains to be experimentally characterized. State-of-the-art bioinformatics tools confirm earlier predictions indicating the presence of an integral membrane helical hairpin, and further suggest anchoring of this protein to the ER membrane, with its N- and C-terminal ends facing the lumen of the organelle. Our evolutionary analysis confirmed the important role of purifying selection in the preservation of the identified domines during the evolution of B169L in nature. Here, we address the possible function of this hairpin transmembrane domain (HTMD) as a class IIA viroporin. Expression of GFP fusion proteins in the absence of a signal peptide supported B169L insertion into the ER as a Type III membrane protein and the formation of oligomers therein. Overlapping peptides that spanned the B169L HTMD were reconstituted into ER-like membranes and the adopted structures analyzed by infrared spectroscopy. Consistent with the predictions, B169L transmembrane sequences adopted canonical alpha-helical conformations in lipid bilayers. Moreover, single vesicle permeability assays demonstrated the assembly of lytic pores in ER-like membranes by B169L transmembrane helices, a capacity confirmed by ion-channel activity measurements in supported bilayers. Emphasizing the relevance of these observations, pore-forming activities were not observed in the case of transmembrane helices derived from EP84R, another ASFV protein predicted to anchor to membranes through a alpha-helical HTMD. Overall, results in this work support the viroporin-like function for the B169L HTMD.